Fig. 5. Shear stress enhances the endothelial barrier through the Sox18 mediated increase in Claudin-5 in pulmonary artery endothelial cells.
PAEC were transfected with either Scrambled siRNA or Claudin-5 siRNA for 24–72 h. Immunoblot analysis indicated a decrease in Claudin-5 protein levels after 48 h and 72 h of transfection (A) Under static conditions, the knockdown of Claudin-5 did not alter endothelial barrier function, as determined by the normalized transendothelial resistance (TER) (B) and by the formation of intercellular gaps (C) indicated by the immunostaining of the tight junction protein, Zonula occludens-1 (ZO-1), using an Alexa Fluor 594 conjugated ZO-1 antibody. No changes in VE-cadherin or stress fiber formation were observed (D). Under shear conditions (20 dyn/cm2, 3h), the silencing of Claudin-5 expression with Claudin-5 siRNA (E) resulted in an immediate decrease in TER compared to scrambled siRNA transfected cells (F). When Claudin-5 was over-expressed in the Claudin-5 depleted cells (G), the increase in barrier function with shear was partially restored (H). Claudin-5 depletion in the presence of shear stress (20 dyn/cm2, 3h) increased intercellular gap formation (I) and disrupted the pattern of VE-cadherin staining (J) but did not alter stress fiber formation (J). Further, immunoblot analysis showed that the exposure of PAEC to Claudin-5 siRNA did not attenuate the shear mediated increase in Sox18 protein levels (K). Values are mean ± SEM, n=3–7. *p<0.05 vs. Scrambled siRNA, Scrambled siRNA+pCMV6 (G-H); † P<0.05 vs. Scrambled siRNA+Shear, Claudin-5 siRNA+pCMV6 (G-H).